Fluorescence Principles and Applications

Photinus pyralis (firefly)

Mycena chlorophos Lobophyllia (meat coral)Glofish

Fluorescence in our surroundings

Now questions in our mind: 1. why they show this property? 2. origin of fluorescence?

• Fluorescence: is the emission of light by a substance that has absorbed light or other electromagnetic radiation.

• Fluorophore:  is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or plane or cyclic molecules with several π bonds.

Absorption of Light

Electronic Excitation

Radiative Mechanism Radiationless Mechanism

Dissipation Mechanism

1. Fluorescence

2. Phosphorescence

Light Light

h h’

Chemical

1. Singlet

2. Triplet

Physical

1. IC

2. ISC

Light Chemical

h ∆GLight Heath Q

Net effect Net effect Net effect

JABLONSKI DIAGRAM

Fluorescence: S1 S0

(10-9-10-6 sec)

Intersystem crossing: S1 T1

(10-11-10-6 sec)

Phosphorescence: T1 S0

(10-3-100 sec)

Variables that affect fluorescence

Fluorescence and the structure:• Simple heterocycles and aromatic

rings do not show fluorescence. • Fusion of benzene ring with

heterocyclic structure results in the fluorescence.

In the native protein Trps (W) are burried Whereas in the unfolded state they are exposed to solvent

• Alkyl branches increases the fluorescence.

• Fluorescence increases as oxygenated species increases in substitution.

• Halogen substitution decreases fluorescence as the molar mass of the halogen increases. 

• In heavy atom substitution such as nitro derivatives or heavy halogen substitution such as iodobenzene, the compounds are subject to predissociation.

• Carboxylic acid or carbonyl group on aromatic ring generally inhibits fluorescence. Therefore, the fluorescence yield from n→π  ∗ transition is low.

VARIABLES THAT AFFECT FLUORESCENCE

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Energy gap is: Less for n-π*High for π-π*

Effect of Structural Rigidity on Fluorescence 

• Fluorescence is particularly favored in molecules with rigid structures.

• This concept of rigidity was used to explain the increase in fluorescence of organic chelating agent when the compound is complexed with a metal ion. The fluorescence intensity of 8-hydroxyquinoline is much less than its zinc complex.

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Temperature and solvent effect• Fluorescence decreases with increasing temperature.

• Fluorescence of a molecule decreases when its solvent contains heavy atoms such as carbon tetrabromide and ethyl iodide, or when heavy atoms are substituted into the fluorescing compound.

PH effectFluorescence of certain compounds have been used as a detection of end points in acid-base titrations. An example of this type of fluorescence seen in compound as a function of pH is the phenolic form of 1-naphthol-4-sulfonic acid

Fluorescein isothiocyanate (FITC)

Fluorescein

FITC

• FITC has excitation and emission spectrum peak wavelengths of approximately 495 nm/519 nm.

• Reactive towards nucleophiles like amine and sulfhydryl groups on proteins

•Useful in drug delivery Studies of anti cancer drugs

Ref: Vineberg et al., Design, Synthesis, and Biological Evaluation of Theranostic Vitamin− Linker−Taxoid Conjugates, J. Med. Chem., ASAP 2015

• When a fluorescent dye (FITC) is conjugated to a monoclonal antibody, it can be used to identify a particular cell type based on the individual antigenic surface markers of the cell. In a mixed population of cells, different fluorochromes can be used to distinguish separate subpopulations. The staining pattern of each subpopulation can be used to identify which cells are present in a sample and to count their relative percentages. The cells can also be sorted if desired.

Fluorescent molecular dyes

Cy3 and Cy5:

• Cyanine dyes are used to label proteins, antibodies, peptides, nucleic acid probes, and any kind of other biomolecules to be used in a variety of fluorescence detection techniques.

• a succinimidyl group to react with amines, or a maleimide group to react with a sulfhydryl group of cysteine residues.•The double bond maleimide readily reacts with the thiol group found on cysteine to form a stable carbon-sulfur bond. maleimideN-hydroxy succinamide

• FRET relies on the distance-dependent transfer of energy from a donor molecule to an acceptor molecule. Due to its sensitivity to distance, FRET has been used to investigate molecular interactions. FRET is the radiationless transmission of energy from a donor molecule to an acceptor molecule. The donor molecule is the dye or chromophore that initially absorbs the energy and the acceptor is the chromophore to which the energy is subsequently transferred.

Fluorescence Resonance Energy Transfer (FRET)

Malachite green

Ref:

• Perhaps the first point to be emphasized is the difference between malachite and malachite green. The former is a copper mineral, CuCO2.Cu(OH)2, while malachite green is an arylmethane dye (C23H5N2) and contains no copper. Specifically, it is a diamino-triphenylmethane with the structure.

• Upon binding of MGA increases the Fluoroscence of the dye >2000 fold

spinach• Spinach is a synthetically derived RNA aptamer born out of the need for a way of studying the role of RNAs at the cellular level.

• It is an 84-nucleotide-long structure with two helical strands and an internal bulge with a G-quadruplex motif, where the fluorophore binds